Project Summary Growth regulation of mammalian cells has been described as One of the last big unsolved problems in cell biology. The ability to measure accurately the growth rate of single cells has been the main obstacle in answering this question. From a clinical perspective, the basic understating of cell growth kinetics and how it is modulated by disease and treatment will allow for more targeted drug development. In recent years, there has been a significant interest in multidisciplinary work by biomedical engineers and scientists with a vision of developing 3D ex vivo tissue models of human organ function, anatomy, and disease. These 3D cellular systems are referred interchangeably as organoid, organotypic, or spheroid (spherical organoid). Organoids self-assemble under proper conditions, i.e., when relevant components, such as extracellular matrix (ECM) proteins, are present. Organoids are well documented to better recapitulate aspects of in vivo organ function and human disease. The common tool for analysis of such systems has been confocal (fluorescence) microscopy of fixed specimens. However, this approach does not reveal structural information in the center of the construct and, most importantly, is limited in terms of time-lapse imaging. There is a critical need for revealing subcellular structures in label-free mode with high contrast, which allows for dynamic, non- destructive imaging. At the same time, quantifying the dry mass of the organoid and its cellular components will inform on the basic organ function and disease, with and without treatment. Despite this critical need, a unified, easy-to-use methodology to measure the growth rate of individual cells and 3D constructs is lacking. Until recently, the state-of-the-art method to assess a single cell growth curve was using Coulter counters to measure the volume of a large number of cells, in combination with careful mathematical analysis. For relatively simple cells such as Escherichia coli (E. coli), traditional microscopy techniques have also been used to assess growth in great detail. In this type of method the assumption is that volume is a good surrogate for mass; however, this assumption is not always valid, for example due to variations in osmotic pressure. We propose to develop a practical dry mass assay for 2D cell populations, as well as 3D organoids, based on a novel imaging method developed in our laboratory: Spatial Light Interference Microscopy (SLIM) for 2D cultures and Gradient Light Interference Microscopy (GLIM) for 3D organoids. SLIM/GLIM takes advantage of the fact that optical phase delay accumulated through a live cell is linearly proportional to the dry mass (non-aqueous content) of the cell. Due to its particular interferometric principle, GLIM significantly suppresses multiple scattering and, as result, is capable of imaging thick specimens such as organoid/spheroids. The project aims to optimize and translate the composite SLIM/GLIM technology into a cell growth assay instrument that can be broadly adopted by researchers in both the research and pharma markets.